Picornaviruses
Picornaviruses are positive-strand RNA viruses that contain a long open reading frame encoding a polyprotein. Cardioviruses and aphthoviruses are two genera of the picornavirus family. The 5'-end untranslated sequences for these genera are typically 750 to 1,300 nucleotides in length. Some strains of cardioviruses and aphthoviruses have a homopolymeric non-coding poly(C) tract which is located about 150 to 330 bases from the 5'-end of the RNA strand.
The length of the poly(C) tract in cardioviruses and aphthoviruses is usually between 60 to 200 bases and the tract may include discontinuities, such as the insertion of a U residue within the stretch of poly(C).
Both the length of the poly(C) tract and the particular discontinuities are characteristics of a particular strain of cardiovirus or aphthovirus. Examples of poly(C) tract-containing cardioviruses are Mengo viruses, EMCV (encephalomyocarditis virus), ME (Maus Elberfeld), Columbia SK, and MM. Foot and mouth disease virus (FMDV) is an example of an aphthovirus containing a poly(C) tract.
Primary Cleavage in Cardioviruses and Aphthoviruses
Mature viral cardioviral and aphthoviral proteins are derived by progressive, post-translational cleavage of the polyprotein that occurs while the peptides are still nascent on ribosomes. (Reviewed by Palmenberg, et al., Ann. Rev. Microbiol. 44:603-623, 1990.) To simplify homolog identification, the European Study Group on the Molecular Biology of Picornaviruses (R. R. Rueckert, et al., J. Virol. 50:957-959, 1984) adopted a uniform nomenclature system, designated L-4-3-4, in 1983. Accordingly, mature picornaviral proteins and their precursors are subdivided into four groups (L, P1, P2, P3) on the basis of structure, enzymatic function, and position of primary cleavages.
The leader or "L" proteins are present only in cardio- and aphthoviruses. The EMCV and Mengo leaders are about 7 kd in molecular weight. FMD viruses have two nested L peptides (16 kd and 23 kd), which share common carboxyl ends, but have different in-phase translational start sites (A. R. Carroll, et al., Nucl. Acids Res. 6:2381-2390, 1984; S. Forss, et al., Nucl. Acids Res. 12:6587-6603, 1984; B. H. Robertson, et al., J. Virol. 54:651-660, 1985).
The four P1 peptides are the capsid structural proteins, VP1, VP2, VP3, and VP4 (1D, 1B, 1C, and 1A), named in order of descending molecular weight on polyacrylamide gels (EMCV: 30, 28, 25 and 8 kd). Protein VP0 (1AB), the uncleaved precursor of VP4+VP2, can also be detected at trace levels in virions.
The middle portion of the polyprotein yields peptides 2A, 2B, and 2C (EMCV: 16, 17, and 36 kd). FMDV genomes have very small or deleted 2A sequences when compared to the other viruses. The biological roles of the P2 peptides are currently under examination. The 2A and 2B components are discussed below in conjunction with their activities in the initial steps of polyprotein processing.
The P3 peptides, 3A, 3B, 3C, and 3D (EMCV: 10, 2, 22, and 51 kd) are more closely associated with genome replication. Purified preparations of 3D can catalyze elongation of nascent RNA chains in primer-dependent reactions, an activity that identifies this enzyme as the central element of viral polymerase complexes (J. B. Flanegan, et al., Proc. Natl. Acad. Sci. USA 74:2677-2680, 1977; J. B. Flanegan, et al., J. Virol. 29:352-360, 1979; R. E. Lundquist, et al., Proc. Natl. Acad. Sci. USA 71:4774-4777, 1974; T. A. Van Dyke, et al., J. Virol. 35:732-740, 1980). Protein 3B is VPg, the peptide attached to the 5'-end of the genome (M. A. Pallansch, et al., J. Virol. 35:414-419, 1980). Protein 3C is a viral-specific protease, responsible for many posttranslational cleavage events (A. E. Gorbalenya, et al., FEBS Lett. 108:1-5, 1979; A. C. Palmenberg, et al. J. Virol. 32:770-778, 1979; Y. V. Svitkin, et al., FEBS Lett. 108:6-9, 1979).
The primary cleavage event within viral polyproteins is co-translational, occurring as soon as a ribosome has reached the middle, or P2 region, of the genome. Distinct processing sites and catalytic mechanisms are used by the various genera. The most thoroughly studied reactions are those of the polio 2A protease, which cleaves its nascent polyprotein at the P1-P2 junction.
A high degree of primary amino acid identity intimates that polio 2A shares its functionality with other members of the enterovirus and rhinovirus genera, but the catalytic sequences are not held in common with the aphthoviruses, cardioviruses, or hepatitis-A viruses. Rather, the cardioviruses (and probably aphthoviruses) seem to achieve efficient primary scission through use of a unique self-cleavage mechanism, dependent on an usually reactive tetrapeptide sequence spanning the 2A-2B junction. The required sequence is not present in hepatitis-A.
Two lines of evidence indicate that cardioviral and aphthoviral 2A peptides are not functionally equivalent to those of the rhino and enteroviruses, and that analogous nascent cleavage activity for these isolates is necessarily located elsewhere in their genomes. First, the 2AB region of aphthoviruses is much shorter than in entero- or rhinoviruses, and sequence comparisons strongly suggest that the missing or deleted segment(s) corresponds to peptide 2A (A. R. Carroll, et al., Nucl. Acids Res. 12:2461-72, 1984; A. C. Palmenberg, in The Molecular Biology of Positive Strand RNA Viruses, ed. D. J. Rolands, B. W. J. Mahy, M. Mayo, pp. 1-15, London: Academic, 1987; B. H. Robertson, et al., J. Virol. 54:651-60, 1985; M. D. Ryan, et al., Virology 173:35-45, 1989).
The second rational for an alternative mechanism is that nascent cleavage within cardioviral and aphthoviral polyproteins actually occurs at a different site than in rhino and enteroviruses (2A-2B versus P1-2A), and releases a much larger primary precursor (L-P1-2A versus P1) (M. J. Grubman, et al., Virology 116:19-30, 1982; D. S. Shih, et al., J. Virol. 30:472-80, 1979). Microsequencing has located the primary site of FMDV-A12 at a G-P sequence, 16 amino acids downstream from the 1D-2A junction (Robertson, et al., J. Virol. 54:651-660, 1985). All other sequenced cardio- and aphthovirus strains maintain a G-P pair in the equivalent position.
Relocation to the G-P site did little to clarify the primary mechanism used by these viruses. Unlike the rhino and enteroviruses, where 2A sequences are recognizable as those of a proteolytic enzyme, extensive pattern searches of cardio- and aphthovirus 2AB segments failed to detect identifiable catalytic motifs (A. Palmenberg, unpublished data). Even more puzzling, cell-free translation experiments showed that large segments of the 2A and 2B coding regions could be entirely deleted from engineered EMCV and FMDV RNA transcripts without affecting cleavage activity in the remaining expressed peptide. The L, P1, 2C, and P3 regions of the genome are likewise dispensable for cleavage activity in extracts from a wide variety of cells (e.g. rabbit reticulocyte, HeLa, wheat germ, and insect) (M. D. Ryan, et al., supra, 1989; A. Palmenberg, unpublished data).
The logical alternatives, that (a) some ubiquitous host protease was responsible for primary scission, or (b) that a small common portion of cardio- and aphthovirus polyproteins was autonomously catalytic, were putatively resolved by the recent creation and testing of synthetic peptide sequences. By focusing on the (relatively) conserved segment spanning the 2A-2B junction, Peter Pallai (personal communication) and colleagues demonstrated that synthetic tetrapeptides containing the viral Asn-Pro-Gly-Pro sequence (N-P-G-P) were spontaneously cleaved to Asn-Pro and Gly-Pro when incubated only in buffer. Although detailed mechanistic studies are still incomplete, the astonishing simple autocatalytic reaction seems to be carried out most efficiently in slightly basic reaction mixtures (i.e. pH 8.0), as might be expected for an authentic physiological event.
Missing in the art of molecular biology is a method of using the autocatalytic cleavage site found in picornaviruses to usefully express a recombinant peptide or protein.